Quantitation of trace protein in lactose for injection using SensiNanoOrange fluorescence assay

Abstract

Lactose is an important pharmaceutical excipient in the drug industry. Residual protein in lactose derived from cow milk can cause acute allergic reactions in some patients, especially when lactose-containing drugs are administered via the intravenous route. The aim of this study was to develop an accurate and reliable method for quantitating trace amount of residual protein in lactose using simple instrumentation. In this work, we developed a new method called SensiNanoOrange that is based on the NanoOrange protein quantitation assay. 1. Compared with the original NanoOrange method, the sample volume was increased from ≤4% to 90% in the final test solution. 2. Using the SensiNanoOrange method, the sensitivity of the test was significantly improved. In this work, an LOQ of 0.6 μg/mL in sample solution was achieved, which is 4.2 times lower than the value reported in the original NanoOrange protocol. The calibration curve was linear from 0.5 to 5 μg/mL. 3. The accuracy and precision of the method for quantitation of protein in lactose solution were confirmed. The results showed that for the accuracy test, all recovery values ranged from 81.45 to 100.85% with RSD ≤ 11.33% in each group. The RSD for the precision test was less than 10.08%. 4. The SensiNanoOrange method was used to indicate that treatment by activated carbon can remarkably reduce the protein content in lactose solution. Our data suggested that this method is not only highly suitable for the characterization of lactose used for injections in the pharmaceutical industry but also may be useful for other biochemical and clinical applications.